Uses of il-12 in hematopoiesis

ABSTRACT

Methods for enhancing or stimulating hematopoiesis including the step of administering Interleukin-12 (IL-12) to yield hematopoietic recovery in a mammal in need. Preferred methods include the step of administering IL-12 as an adjuvant therapy to alleviate the hematopoietic toxicities associated with one or more treatment regimens used to combat a disease state. Other methods include administering IL-12 to ameliorate various hematopoietic deficiencies. Still other methods are directed to uses of IL-12 for in-vivo proliferation of hematopoietic repopulating cells, hematopoietic progenitor cells and hematopoietic stem cells. Other disclosed methods are directed to uses of Il-12 for bone marrow preservation or recovery.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/076,365, filed Mar. 30, 2011, which is a continuation of U.S. patentapplication Ser. No. 10/886,267, filed Jul. 6, 2004, which claimspriority from U.S. Provisional Patent Application No. 60/485,170, filedon Jul. 3, 2003. All of these applications are incorporated herein byreference in their entirety.

BACKGROUND

Many disease states result from insufficient hematopoiesis generally, orinsufficient hematopoiesis in one or more blood cell types or bloodlineages. Still other disease states result from dysfunction in one ormore lineages of the hematopoietic system, such as leukemia. Moreover,many treatments that are given to an individual to combat certaindisease states, such as cancer, incidentally decrease the individual'sblood supply and/or can lead to bone marrow failure. The toxichematopoietic side effects of these treatments, therefore, decrease thelikelihood of the treatment being successful, and often are the limitingeffect that results in cessation of treatment.

Thus, there is a need to develop therapies that can restorehematopoiesis and alleviate hematopoietic deficiencies. Such therapieswould be capable of maintaining, stimulating and/or modulating the bloodsupply, and also would be useful as an ancillary therapy given todecrease the toxic effects of a primary therapy, such as chemotherapy orradiation therapy.

SUMMARY OF THE INVENTION

The present invention is generally directed to uses of Interleukin-12(IL-12) in stimulating or enhancing hematopoiesis in a subject in needthereof. In the various embodiments of the invention, the subject is amammal, and the preferred mammal is a human. The preferred human dosagesof IL-12 suitable for use in the various embodiments of the inventionrange from about 2000 ng/kg to less than about 10 ng/kg, however,dosages of about 1 ng/kg or less may also be suitable in particulartherapeutic methods of the present invention.

In the embodiments of the present invention, hematopoiesis can involvein-vivo proliferation or expansion of hematopoietic repopulating cell,hematopoietic stem/progenitor cells or other hematopoietic cells,generally residing in bone marrow, or hematopoiesis can involveprotection of hematopoietic repopulating cells, stem/progenitor cells orother hematopoietic cells, generally residing in bone marrow, fromchemical and/or radiation injury.

An embodiment of the present invention is directed to methods fortreating a disease state in a mammal. The method includes administeringa treatment to the mammal that is intended to target the disease state,where the treatment has an associated hematopoietic toxicity, andadditionally administering one or more therapeutically effective dose(s)of IL-12 near the time of administration of the treatment, where theadministration of IL-12 to the mammal reduces the hematopoietic toxicityof the treatment. Administration of IL-12 in this embodiment of theinvention, can take place either before, after or before and after,administration of the treatment intended to target the disease state.

In this embodiment of the present invention, the treatment that isintended to target the disease state is either a form of chemotherapy orradiation therapy, or both. Additionally, the treatment intended totarget the disease state generally results in a deficiency in one ormore hematopoietic cell types or lineages, which is this embodiment ofthe invention is ameliorated by administration of IL-12. Moreover, inthis embodiment of the invention, high dose treatment modalities, suchas near lethal treatment modalities of a chemotherapy and/or radiationtherapy, and dose dense treatment regimens can be utilized and more thanone type of chemotherapy or radiation therapy can be administered, thusgreatly facilitating the eradication or suppression of the diseasestate. Also in the embodiments of the invention, one or moretherapeutically effective dose(s) of IL-12 can administered at varioustime intervals before, before and after, or after the administration ofthe treatment.

Various disease states can be treated by this embodiment of theinvention. For example, the disease state can be manifested in a form ofcancer, where the treatment includes a form of chemotherapy or radiationtherapy and is targeted to treating one or more types of solid tumors,such as tumors manifested in breast, lung, prostate, ovarian cancer, orthe like. Other forms of cancers that are included in this embodiment ofthe invention are hematopoietic cell cancers, such as leukemias orlymphomas or the like. In either the case where the disease state ismanifested by the presentation of solid tumor or hematopoietic celldisorders (cancers), practicing this method embodiment of the presentinvention can result in increases rate of remission of the tumors, ascompared with administering the treatment modality that is intended totarget the disease state alone.

Generally in this embodiment of the invention, the administration ofIL-12 results in protection of bone marrow cells from the associatedhematopoietic toxicity of the treatment, such as chemoprotection of bonemarrow cells when the treatment includes chemotherapy andradioprotection of bone marrow cells when the treatment includesradiation therapy. Further, in this embodiment bone marrow cells caninclude hematopoietic repopulating cells, hematopoietic stem cells orhematopoeitic progenitor cells.

Another embodiment of the present invention is directed to methods oftreating a mammal for a deficiency in hematopoiesis by administering oneor more therapeutically effective dose(s) of IL-12 as needed toameliorate the deficiency. This deficiency in hematopoiesis may be ageneral deficiency or may include one or more deficiency in specifichematopoietic cell lineages or cell types. In this embodiment of theinvention, the deficiency may be exacerbated upon the administration ofvarious forms of chemotherapy or radiation therapy. Moreover, thedeficiency may substantially be the result of a disease state that ismanifested in the mammal.

In this embodiment, the deficiency is ameliorated by the IL-12facilitated proliferation of one or more types of bone marrow cells.Further, in this embodiment, the deficiency is preferentiallyameliorated by the IL-12 facilitated proliferation of hematopoieticrepopulating cells, hematopoietic stem cells or hematopoietic progenitorcells.

In this embodiment of the invention, as stated above, the deficiency maybe a general deficiency in hematopoiesis in the mammal, or thedeficiency may include a deficiency in one or more specifichematopoietic cell lineages, such as a low white blood cell count, redblood cell count, platelet count, neutrophil count, monocyte count,lymphocyte count, granulocyte count, dendritic cell count, or the like.These deficient states of hematopoiesis may be characterized as alymphopenia, myelopenia, leukopenia, neutropenia, erythropenia,megakaryopenia, or the like.

Also in this embodiment of the invention, various underlying diseasestates may be responsible for the hematopoietic deficiency, includingvarious forms of cancer, or other disease states listed in Table Ibelow.

Another embodiment of the present invention is directed to methods ofstimulating or enhancing hematopoiesis in a mammal by administering oneor more therapeutically effective dose(s) of IL-12 for a duration thatis effective to achieve a therapeutic result that includes thestimulation or enhancement of hematopoiesis. In this embodiment, thestimulation or enhancement of hematopoiesis involves the 11-12facilitated proliferation of bone marrow cells, preferentially, thestimulation or enhancement of hematopoiesis involves the IL-12facilitated proliferation of hematopoietic repopulating cells, includinglong-term repopulating cells, hematopoietic progenitor cells orhematopoietic stem cells. Also included in this embodiment are methodsthat also utilize the administration of radiation therapy and/orchemotherapy.

In this embodiment of the invention, as stated above, the deficiency maybe a general deficiency in hematopoiesis in the mammal, or thedeficiency may include a deficiency in one or more specifichematopoietic cell lineages, such as a low white blood cell count, redblood cell count, platelet count, neutrophil count, monocyte count,lymphocyte count, granulocyte count, dendritic cell count, or the like.These deficient states of hematopoiesis may be characterized as alymphopenia, myelopenia, leukopenia, neutropenia, erythropenia,megakaryopenia, or the like.

Also in this embodiment of the invention, various underlying diseasestates may be responsible for the hematopoietic deficiency, includingvarious forms of cancer, or other disease states listed in Table Ibelow.

Still another embodiment of the present invention is directed to methodsfor bone marrow preservation or recovery in a mammal that involveadministering one or more therapeutically effective dose(s) of IL-12 tothe mammal, without the use of hematopoietic repopulating cells,hematopoietic progenitor cells or hematopoietic stem cells, for aduration necessary for bone marrow preservation or recovery. Thesemethods are suitable for use to counteract the effects of bone marrowfailure or when the mammal is suffering from a disease state and neardestruction of the bone marrow is a by-product of a treatment regimenrecommended to combat the disease state. These methods of the inventionare useful in that the need for a bone marrow transplant may beobviated, thus eliminating the generally negative side effects of suchtransplants whether they be allogenic or autologous.

These methods include bone marrow preservation or recovery that involvesincrease in hematopoietic repopulating cell, hematopoietic stem cells orhematopoietic progenitor cells, an increase in one or moredifferentiated hematopoietic cells types and/or an increase inhematopoietic support cells.

In this embodiment of the invention, as stated above, the deficiency maybe a general deficiency in hematopoiesis in the mammal, or thedeficiency may include a deficiency in one or more specifichematopoietic cell lineages, such as a low white blood cell count, redblood cell count, platelet count, neutrophil count, monocyte count,lymphocyte count, granulocyte count, dendritic cell count, or the like.These deficient states of hematopoiesis may be characterized as alymphopenia, myelopenia, leukopenia, neutropenia, erythropenia,megakaryopenia, or the like.

Also in this embodiment of the invention, various underlying diseasestates may be responsible for the hematopoietic deficiency, includingvarious forms of cancer, or other disease states listed in Table Ibelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a Kaplan-Meier survival curve for the radioprotectiveeffect of IL-12 administration; the data show that IL-12 protects micefrom lethal ionizing radiation.

FIGS. 2A-C shows that IL-12 administration protects bone marrow fromlethal dose radiation in animal models. At different time post IL-12treatment (5 ug/kg) and lethal dose radiation (1, 12 and 14 days),femurs were isolated, fixed in 10% formalin, decalcified and processedfor paraffin section and for Hematoxylin & Eosin staining. FIG. 2A: 1day post radiation; FIG. 2B: 12 days post radiation; FIG. 2C: 14 dayspost radiation. Although there is no significant histologicaldifferences at the time points of day 1, 7, and 10 (results were notshown for day 7 and 10), colonies were observed at day 12 in the micetreated with IL-12 (FIG. 2B). At day 14, hone marrow in IL-12 treatedmice shows significant regeneration while there is not obviousregeneration in control mice bone marrow. (200× magnification)

FIG. 3 show that low dose IL-12 (5 ug/kg) does not sensitizegastro-intestinal tract to two lethal dose radiation in animal models;IL-12 treated (5 ug/kg (approximately 10 ng/mouse)) or control micereceived different dose radiation (10 and 16 Gy for bone marrow and GItract lethal dose respectively). 4 days post radiation, small intestinewere removed, fixed for PAS staining for mice received 10 Gy (PeriodicAcid Schiff), H&E staining for mice received 16 Gy. There is nosignificant histological difference between control and IL-12 treatedmice with both radiation dose. There is no obvious histological damagein mice who received 10 Gy radiation. (200× magnification)

FIGS. 4A-4F shows that IL-12 administration promotes multiple lineageblood cell recovery of sublethally irradiated mice; After IL-12treatment (100 ng/mouse) and sublethal radiation (500 rad), peripheralblood were collected at different time via tail vein for blood cellcounting and differentiation (Mascot from Brew). FIG. 4A: white bloodcell count; FIG. 4B: red blood cell count; FIG. 4C: platelet count; FIG.4D: lymphocyte; FIG. 4E: monocyte; FIG. 4F: neurtrophile. Blue is thecontrol while pink is IL-12 treatment 24 hours before irradiation andred is IL-12 treatment 1 hour after irradiation. IR: irradiation. pvalue for each subtype cell is under the figure and those p values lessthan 0.05 or 0.001 are in red color.

FIGS. 5A-5F IL-12 did not protect bone marrow progenitor cells orshort-term hematopoietic stem cells (ST-HSCs) from lethal irradiationsince there were no or little CFC (FIG. 5A) and CFU (FIG. 5B) activitiesin bone marrow cells immediately after or 7 days after irradiation. Till10 days for CFU (FIGS. 5B&C) or 14 days for CFC (FIG. 5A) afterirradiation, the colony-forming activities are almost fully recovered.These later CFU and CFC activities indicate they both were derived fromprotected long-term hematopoietic stem cells (LT-HSCs). The recovery forCFU (ST-HSCs) was about 4 days earlier than CFC (bone marrow progenitorcells). This matches with the normal bone marrow stem cellsdifferentiation steps beginning from LT-HSC to ST-HSCs, then to morecommitted progenitor cells. Active LT-HSCs not only rescue lethallyirradiated mice but also can completely repopulate the hematopoieticsystem. IL-12-protected LT-HSCs not only rescued the lethally irradiatedmice and repopulated the hematopoietic system (FIG. 5F) but alsodifferentiated into functional myeloid lineage (FIG. 5D) and lymphoidlineage (FIG. 5E) in long term. IL-12 administration protects long-termrepopulating stem cells in lethally irradiated mice; At different timepost lethal dose radiation, bone marrow cells were isolated from micewho received IL-12 or PBS buffer for colony forming cell assay (CFCassay) (FIG. 5A), colony forming units spleen day 12 assay (CFU-512)(FIG. 5B-5C), and bone marrow transplantation (BMT, FIG. 5D-F); D: daysafter irradiation, D0: immediately after irradiation; IR, irradiation.BM, bone marrow. CFU-S₁₂, colony forming units—spleen12.

FIG. 6 shows that IL-12 administration promotes multiple lineage bloodcell recovery of lethally irradiated mice; After IL-12 treatment (100ng/mouse) and lethal dose radiation (10 Gy), peripheral blood werecollected at different time via tail vein for blood cell counting anddifferentiation (Mascot from Brew). FIG. 6A: white blood cell count;FIG. 6B: red blood cell count; FIG. 6C: platelet count. By day 14 postradiation, all the mice from control group died, but the mice receivedIL-12 started to recover.

FIG. 7 shows that IL-12 administration generally stimulates bone marrowcell proliferation; 24 hours post IL-12 administration (10 ng/mouse),bone marrow cells were isolated for BrdU incooperation assay (FIG. 7A).Statistic analysis showed IL-12 treated bone marrow contains higher % ofBrdU positive cells compared with control mouse (B, p<0.01, n=6). Cellcycle analysis were performed with Propidium Iodide staining methodwhich showed significant increased cell number at S phase (C, p<0.05,n=6).

FIG. 8 IL-12 could significantly increase the percentage of Annexin Vnegative/Sca-1 positive cells (active bone marrow stem cells) in wholebone marrow compared with that of control (FIG. 8: H₄ 19.2±2.3% VS9.7±1.5%, p<0.01) after irradiation. The proportion of Annexin Vnegative/Sca-1 positive cells in total Sca-1 positive cells (FIG. 8:H₂+H₄) was also significantly higher for IL-12 treatment than control(54±5% vs 44±5%, p<0.05). It indicated that there were more active bonemarrow cells with stem markers (such as Sca-1) in IL-12 treated miceafter lethal irradiation. IL-12 administration protects AnnexinV⁻/Sca-1⁺ cells (H₄) from radiation induced apoptosis, where the Sca-1⁺positive cells are indicative of the presence of hematopoieticrepopulating cell or hematopoietic stem cells and Annexin V negativecells are indicative of the functional bone marrow cells.

FIGS. 9A-9F shows that IL-12 administration promotes multiple lineageblood cell recovery from the hematopoietic insult of chemotherapeuticdrugs; IL-12 was administrated at different time (36 hrs before or 12hrs after cytoxan) to mice who received relatively high doses of thechemotherapeutic drug Cytoxan (300 mg/kg). FIG. 9A: white blood cell;FIG. 9B: red blood cell; FIG. 9C: platelet; FIG. 9D: lymphocyte; FIG.9E: monocyte; FIG. 9F: neutrophile. Cytoxan is name forcyclophosphamide.

DESCRIPTION OF THE INVENTION

Embodiments of the present invention provide for various methods ofusing Interleukin-12 (IL-12) in hematopoiesis, particular forstimulating or enhancing hematopoiesis in a mammal in need. The presentinvention includes various approaches to using IL-12 to increase theproduction of hematopoietic cell types or lineages.

A particular embodiment of the present invention is directed to treatinga disease state in a mammal, where treatment modalities themselvesgenerally produce decreases or deficiencies in hematopoiesis as alimiting toxicity to the treatment modality. Other embodiments of theinvention provide for methods of generally treating a mammal for adeficiency in one or more hematopoietic cell types or lineages, methodsfor stimulating or enhancing hematopoiesis and methods of preservationand recovery cells that comprise bone marrow.

An indication of the therapeutic effectiveness of the therapeuticmethods of the present invention is demonstrated by the ability of thesemethods to confer survival on lethally irradiated animals as illustratedbelow. Recovery from lethal irradiation provides an extreme indicationof the therapeutic effectiveness of the methods of the presentinvention. However, as shown in the examples below, the therapeuticmethods of the present invention can promote hematopoiesis andhematopoietic recovery in the face of less than lethal doses ofirradiation, or less than lethal doses of chemotherapeutic agents, or indisease states where increased hematopoiesis is of therapeutic benefit.

The first embodiment of the methods of the invention involves treatingthe mammal for a generally treatment-induced deficiency inhematopoiesis. The second embodiment of the methods of the inventioninvolves treating the mammal for a deficiency in hematopoiesis where thedeficiency is substantially the result of a disease state, but may beexacerbated by treatment modalities. Both embodiments of the inventionprovide for enhanced hematopoiesis and/or hematopoietic recovery byadministering interleukin-12, or a substantial equivalent, to themammal.

Other embodiments of the present invention provide methods of usingIL-12 to promote, stimulate or enhance hematopoiesis and/orhematopoietic recovery in mammals suffering from a deficiency or defectin hematopoiesis. Still other embodiments provide for preservation orrecovery of bone marrow by administering IL-12 in accordance with themethods provided herein. Moreover, in all embodiments of the invention,IL-12 promoted, stimulated or enhanced hematopoiesis appears to belargely generated from the level of the hematopoietic repopulating,hematopoietic stem or hematopoietic progenitor cell compartment.Further, the uses of IL-12 in embodiments of the invention areparticularly useful as methods of treatment in the medical fields ofoncology and hematology.

DEFINITIONS

The following definitions are provided to give clarity to language usedwithin the specification; language used to clarify definitions is meantto be interpreted broadly and generically.

“Disease state” refers to a condition present in a mammal whereby thehealth and well being of the mammal is compromised. In certainembodiments of the invention, treatments intended to target the diseasestate are administered to the mammal.

“A treatment” is intended to target the disease state and combat it,i.e., ameliorate the disease state. The particular treatment thus willdepend on the disease state to be targeted and the current or futurestate of medicinal therapies and therapeutic approaches. A treatment mayhave associated toxicities.

“An associated hematopoietic toxicity” is a toxicity that substantiallyarises from the administration of the treatment to a mammal thatadversely affects the hematopoietic system of the mammal. This adverseeffect can be manifested in the mammal broadly whereby manyhematopoietic cell types are altered from what is considered to benormal levels, as a result of the treatment, or as a result of thetreatment and the disease state combined, or the adverse effect can bemanifested in the mammal more specifically whereby only one or a fewhematopoietic cell types are altered from what is considered to benormal levels, as a result of the treatment, or as a result of thetreatment and the disease state combined.

“Interleukin-12 (IL-12)” refers to any IL-12 molecule that yields atleast one of the hematopoietic properties disclosed herein, includingnative IL-12 molecules, variant 11-12 molecules and covalently modifiedIL-12 molecules, now known or to be developed in the future, produced inany manner known in the art now or to be developed in the future.Generally, the amino acid sequences of the IL-12 molecule used inembodiments of the invention are derived from the specific mammal to betreated by the methods of the invention. Thus, for the sake ofillustration, for humans, generally human IL-12, or recombinant humanIL-12, would be administered to a human in the methods of the invention,and similarly, for felines, for example, the feline IL-12, orrecombinant feline IL-12, would be administered to a feline in themethods of the invention. Also included in the invention, however, arecertain embodiments where the IL-12 molecule does not derive its aminoacid sequence from the mammal that is the subject of the therapeuticmethods of the invention. For the sake of illustration, human IL-12 orrecombinant human IL-12 may be utilized in a feline mammal. Still otherembodiments of the invention include IL-12 molecules where the nativeamino acid sequence of IL-12 is altered from the native sequence, butthe IL-12 molecule functions to yield the hematopoietic properties ofIL-12 that are disclosed herein. Alterations from the native,species-specific amino acid sequence of IL-12 include changes in theprimary sequence of IL-12 and encompass deletions and additions to theprimary amino acid sequence to yield variant IL-12 molecules. An exampleof a highly derivatized IL-12 molecule is the redesigned IL-12 moleculeproduced by Maxygen, Inc. (Leong S R, et al., Proc Natl Acad Sci USA.2003 Feb. 4; 100 (3): 1163-8.), where the variant IL-12 molecule isproduced by a DNA shuffling method. Also included are modified IL-12molecules are also included in the methods of invention, such ascovalent modifications to the IL-12 molecule that increase its shelflife, half-life, potency, solubility, delivery, etc., additions ofpolyethylene glycol groups, polypropylene glycol, etc., in the mannerset forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337. One type of covalent modification of the IL-12molecule is introduced into the molecule by reacting targeted amino acidresidues of the IL-12 polypeptide with an organic derivatizing agentthat is capable of reacting with selected side chains or the N- orC-terminal residues of the IL-12 polypeptide. Both native sequence IL-12and amino acid sequence variants of IL-12 may be covalently modified.Also as referred to herein, the IL-12 molecule can be produced byvarious methods known in the art, including recombinant methods. Sinceit is often difficult to predict in advance the characteristics of avariant IL-12 polypeptide, it will be appreciated that some screening ofthe recovered variant will be needed to select the optimal variant. Apreferred method of assessing a change in the hematological stimulatingor enhancing properties of variant IL-12 molecules is via the lethalirradiation rescue protocol disclosed below. Other potentialmodifications of protein or polypeptide properties such as redox orthermal stability, hydrophobicity, susceptibility to proteolyticdegradation, or the tendency to aggregate with carriers or intomultimers are assayed by methods well known in the art.

“One or more therapeutically effective dose(s) of IL-12” is any doseadministered for any time intervals and for any duration that cansubstantially ameliorate either the associated hematopoietic toxicity ofthe treatment intended to target the disease state and/or cansubstantially ameliorate a hematopoietic deficiency in one or morehematopoietic cell types, or is capable of stimulating hematopoiesis bystimulating or enhancing the proliferation of hematopoietic repopulatingcells, hematopoietic progenitor cells or hematopoietic stem cells.

“Near the time of administration of the treatment” refers to theadministration of IL-12 at any reasonable time period either beforeand/or after the administration of the treatment, such as one month,three weeks, two weeks, one week, several days, one day, 20 hours,several hours, one hour or minutes. Near the time of administration ofthe treatment may also refer to either the simultaneous or nearsimultaneous administration of the treatment and IL-12, i.e. withinminutes to one day.

“Chemotherapy” refers to any therapy that includes natural or syntheticagents now known or to be developed in the medical arts. Examples ofchemotherapy include the numerous cancer drugs that are currentlyavailable. However, chemotherapy also includes any drug, natural orsynthetic, that is intended to treat a disease state. In certainembodiments of the invention, chemotherapy may include theadministration of several state of the art drugs intended to treat thedisease state. Examples include combined chemotherapy with docetaxel,cisplatin, and 5-fluorouracil for patients with locally advancedsquamous cell carcinoma of the head (Tsukuda, M. et al., Int J ClinOncol. 2004 June; 9 (3): 161-6), and fludarabine and bendamustine inrefractory and relapsed indolent lymphoma (Konigsmann M, et al., LeukLymphoma. 2004; 45(9): 1821-1827).

“Radiation therapy” refers to any therapy where any form of radiation isused to treat the disease state. The instruments that produce theradiation for the radiation therapy are either those instrumentscurrently available or to be available in the future.

“High dose treatment modalities” refer to treatments that are highsub-lethal or near lethal. High dose treatment modalities are intendedto have an increased ability to combat a disease state, but generallypossess increased associated toxicities. Further, generally high dosetreatment modalities exhibit increased hematopoietic toxicities, ascompared with conventional treatment modalities. The protocols for highdose treatment modalities are those currently used or to be used in thefuture.

“A dose dense treatment regimen” is generally a treatment regimenwhereby the treatment is repeated sequentially in an accelerated mannerto achieve the desired treatment outcome, as compared with conventionaltreatment regimens. The methods of the invention facilitate the use ofdose dense treatment regimens by reducing or ameliorating the associatedhematopoietic toxicities of the treatment, thereby permitting dose densetreatment regimens to be utilized and increasing the rate of success intreating a particular disease state. (see generally, Hudis C A, SchmitsN, Semin Oncol. 2004 June; 31 (3 Suppl 8): 19-26; Keith B et al., J ClinOncol. 2004 Feb. 15; 22 (4): 749; author reply 751-3; Maurel J et al,Cancer. 2004 Apr. 1; 100 (7): 1498-506; Atkins C D, J Clin Oncol. 2004Feb. 15; 22 (4): 749-50.)

“Chemoprotection or radioprotection” refers to protection from, or anapparent decrease in, the associated hematopoietic toxicity of atreatment intended to target the disease state.

“An increased remission” refers to a decrease, lessening, reduction,shrinking, diminution, or the like in one or more measurable parametersof a particular disease state.

“Solid tumors” generally refers to the presence of cancer of bodytissues other than blood, bone marrow, or the lymphatic system.

“Hematopoietic disorders (cancers)” generally refers to the presence ofcancerous cells originated from hematopoietic system.

“Ameliorate the deficiency” refers to a reduction in the hematopoieticdeficiency, i.e., an improvement in the deficiency, or a restoration,partially or complete, of the normal state as defined by currentlymedical practice. Thus, amelioration of the hematopoietic deficiencyrefers to an increase in, a stimulation, an enhancement or promotion of,hematopoiesis generally or specifically. Amelioration of thehematopoietic deficiency can be observed to be general, i.e., toincrease two or more hematopoietic cell types or lineages, or specific,i.e., to increase one hematopoietic cell type or lineages.

“Bone marrow cells” generally refers to cells that reside in and/or hometo the bone marrow compartment of a mammal. Included in the term “bonemarrow cells” is not only cells of hematopoietic origin, including butnot limited to hematopoietic repopulating cells, hematopoietic stem celland/or progenitor cells, but any cells that may be derived from bonemarrow, such as endothelial cells, mesenchymal cells, bone cells, neuralcells, supporting cells (stromal cells), including but not limited tothe associated stem and/or progenitor cells for these and other celltypes and lineages.

“Hematopoietic cell type” generally refers to differentiatedhematopoietic cells of various types, but can also include thehematopoietic progenitor cells from which the particular hematopoieticcell types originate from, such as various blast cells referring to allthe cell types related to blood cell production, including stem cells,progenitor cells, and various lineage cells, such as myeloid cells,lymphoid cell, etc.

“Hematopoietic cell lineage” generally refers to a particular lineage ofdifferentiated hematopoietic cells, such as myeloid or lymphoid, butcould also refer to more differentiated lineages such as dendritic,erythroid, etc.

“IL-12 facilitated proliferation” of cells refers to an increase, astimulation, or an enhancement of hematopoiesis that at least partiallyattributed to an expansion, or increase, in cells that generally resideor home to the bone marrow of a mammal, such as hematopoietic progenitorand/or stem cells, but includes other cells that comprise themicroenvironment of the bone marrow niche.

“Stimulation or enhancement of hematopoiesis” generally refers to anincrease in one or more hematopoietic cell types or lineages, andespecially relates to a stimulation or enhancement of one or morehematopoietic cell types or lineages in cases where a mammal has adeficiency in one or more hematopoietic cell types or lineages.

“Hematopoietic long-term repopulating cells” are generally the mostprimitive blood cells in the bone marrow; they are the blood stem cellsthat are responsible for providing life-long production of the variousblood cell types and lineages.

“Hematopoietic stem cells” are generally the blood stem cells; there aretwo types: “long-term repopulating” as defined above, and “short-termrepopulating” which can produce “progenitor cells” for a short period(weeks, months or even sometimes years depending on the mammal).

“Hematopoietic progenitor cells” are generally the first cells todifferentiate from (i.e., mature from) blood stem cells; they thendifferentiate (mature) into the various blood cell types and lineages.

“Hematopoietic support cells” are the non-blood cells of the bonemarrow; these cells provide “support” for blood cell production. Thesecells are also referred to as bone marrow stromal cells.

“Bone marrow preservation” means the process whereby bone marrow thathas been damaged by radiation, chemotherapy, disease or toxins ismaintained at its normal, or near normal, state; “bone marrow recovery”means the process whereby bone marrow that has been damaged byradiation, chemotherapy, disease or toxins is restored to its normal,near normal state, or where any measurable improvement in bone marrowfunction are obtained; bone marrow function is the process wherebyappropriate levels of the various blood cell types or lineages areproduced from the hematopoietic (blood) stem cells.

“Bone marrow failure” is the pathologic process where bone marrow thathas been damaged by radiation, chemotherapy, disease or toxins is notable to be restored to normal and, therefore, fails to producesufficient blood cells to maintain proper hematopoiesis in the mammal.

Hematopoietic Cell Production:

See generally, U.S. Pat. Nos. 5,968,513, 5,851,984 and 6,159,462. Themorphologically recognizable and functionally capable cells circulatingin blood include erythrocytes, macrophage or monocyte, neutrophilic,eosinophilic, and basophilic granulocytes, B-, T, non B-, nonT-lymphocytes, and platelets. These mature hematopoietic cells derivefrom and are replaced, on demand, by morphologically recognizabledividing precursor cells for the respective lineages such aserythroblasts for the erythrocyte series, myeloblasts, promyelocytes andmyelocytes for the monotyte/macrophage and granulocyte series, andmegakaryocytes for the platelets.

The precursor cells derive from more primitive cells that cansimplistically be divided into two major subgroups: stem cells andprogenitor cells. The definitions of stem and progenitor cells areoperational and depend on functional, rather than on morphological,criteria. Stem cells have extensive self-renewal or self-maintenancecapacity, a necessity since an absence or depletion of these cells couldresult in the complete depletion of one or more cell lineages or celltypes, events that would lead within a short time to disease and death.Some of the stern cells differentiate upon need, but some stem cells ortheir daughter cells produce other stem cells to maintain the preciouspool of these cells. Thus, in addition to maintaining their own kind,pluripotential stem cells, or hematopoietic repopulating cells, arecapable of differentiation into several sub-lines of progenitor cellswith more limited self-renewal capacity or no self-renewal capacity.These progenitor cells ultimately give rise to the morphologicallyrecognizable precursor cells. The progenitor cells are capable ofproliferating and differentiating along one, or more than one, of themyeloid differentiation pathways (Lajtha, L. G. (Rapporteur), 1979,Blood Cells 5: 447).

Additionally, chemotherapy and radiation therapy used in the treatmentof cancer and certain immunological disorders can cause pancytopenias orcombinations of anemia, neutropenia and thrombocytopenia. Thus, theincrease or replacement of hematopoietic cells is often crucial to thesuccess of such treatments. (For a general discussion of hematologicaldisorders and their causes, see, e.g., “Hematology” in ScientificAmerican Medicine, E. Rubenstein and D. Federman, eds., Volume 2,chapter 5, Scientific American, New York (1996)).

Furthermore, aplastic anemia presents a serious clinical condition asthe overall mortality of all patients with aplastic anemias, in theabsence of stem cell therapy, is high. Approximately 60-75% ofindividuals suffering from the disorder die within 12 months, in theabsence of new stem cells. The overall incidence of these diseases isapproximately 25 new cases per million persons per year. Although it isextremely unlikely that a single pathogenic mechanism accounts for allaplastic anemias, it is clear that provision of new hematopoietic stemcells is usually sufficient to allow permanent recovery, sincetransplantation of patients with aplastic anemia with bone marrowobtained from identical twins (i.e., syngeneic) (Pillow, R. P., et al.,1966, N. Engl. J. Med. 275: 94-97) or from HLA-identical siblings (i.e.,allogeneic) (Thomas, E. D., et al., Feb. 5, 1972, The Lancet, pp.284-289) can fully correct the disease. However, some patients withaplastic anemia reject the transplanted marrow. This complication isparticularly common among patients who have been immunologicallysensitized as a result of multiple therapeutic blood transfusions.

The current therapy available for many hematological disorders as wellas the destruction of the endogenous hematopoietic cells caused bychemotherapy or radiotherapy is bone marrow transplantation. However,use of bone marrow transplantation is severely restricted since it isextremely rare to have perfectly matched (genetically identical) donors,except in cases where an identical twin is available or where bonemarrow cells of a patient in remission are stored in a viable frozenstate. Except in such autologous cases, there is an inevitable geneticmismatch of some degree, which entails serious and sometimes lethalcomplications. These complications are two-fold. First, the patient isusually immunologically incapacitated by drugs beforehand, in order toavoid immune rejection of the foreign bone marrow cells (host versusgraft reaction). Second, when and if the donated bone marrow cellsbecome established, they can attack the patient (graft versus hostdisease), who is recognized as foreign. Even with closely matched familydonors, these complications of partial mismatching are the cause ofsubstantial mortality and morbidity directly due to bone marrowtransplantation from a genetically different individual.

Peripheral blood has also been investigated as a source of repopulatingcells, or stem cells for hematopoietic reconstitution (Nothdurtt, W., etal., 1977, Scand. J. Haematol. 19: 470-481; Sarpel, S. C., et al., 1979,Exp. Hematol. 7: 113-120; Ragharachar, A., et al., 1983, J. Cell.Biochem. Suppl. 7A: 78; Juttner, C. A., et al., 1985, Brit. J. Haematol.61: 739-745; Abrams, R. A., et al., 1983, J. Cell. Biochem. Suppl. 7A:53; Prummer, O., et al., 1985, Exp. Hematol. 13: 891-898). In somestudies, promising results have been obtained for patients with variousleukemias (Reiffers, J., et al., 1986, Exp. Hematol. 14: 312-315;Goldman, J. M., et al., 1980, Br. J. Haematol. 45: 223-231; Tilly, H.,et al., Jul. 19, 1986, The Lancet, pp. 154-155; see also To, L. B. andJuttner, C. A., 1987, Brit. J. Haematol. 66: 285-288, and referencescited therein); and with lymphoma (Korbling, M., et al., 1986, Blood 67:529-532). Other studies using peripheral blood, however, have failed toeffect reconstitution (Hershko, C., et al., 1979, The Lancet 1: 945-947;Ochs, H. D., et al., 1981, Pediatr. Res. 15: 601). Studies have alsoinvestigated the use of fetal liver cell transplantation (Cain, G. R.,et al., 1986, Transplantation 41: 32-25; Ochs, H. D., et al., 1981,Pediatr. Res. 15: 601; Paige, C. J., et al., 1981, J. Exp. Med. 153:154-165; Touraine, J. L., 1980, Excerpta Med. 514: 277; Touraine, J. L.,1983, Birth Defects 19: 139; see also Good, R. A., et al., 1983,Cellular Immunol. 82: 44-45 and references cited therein) or neonatalspleen cell transplantation (Yunis, E. J., et al., 1974, Proc. Natl.Acad. Sci. U.S.A. 72: 4100) as stem cell sources for hematopoieticreconstitution. Cells of neonatal thymus have also been transplanted inimmune reconstitution experiments (Vickery, A. C., et al., 1983, J.Parasitol. 69 (3): 478-485; Hirokawa, K., et al., 1982, Clin. Immunol.Immunopathol. 22: 297-304).

Interleukin-12 (IL-12)

For general descriptions relating IL-12, see U.S. Pat. Nos. 5,573,764,5,648,072, 5,648,467, 5,744,132, 5,756,085, 5,853,714 and 6,683,046.Interleukin-12 (IL-12) is a heterodimeric cytokine generally describedas a proinflamatory cytokine that regulates the activity of cellsinvolved in the immune response (Fitz K M, et al., 1989, J. Exp. Med.170: 827-45). Generally IL-12 stimulates the production of interferon-γ(INF-γ) from natural killer (NK) cells and T cells (Lertmemongkolchai G,Cai et al., 2001, Journal of Immunology. 166: 1097-105; Cui J, Shin T,et al., 1997, Science. 278: 1623-6; Ohteki T, Fukao T, et alk., 1999, J.Exp. Med. 189: 1981-6; Airoldi I, Gri G, et al., 2000, Journal ofImmunology. 165: 6880-8), favors the differentiation of T helper 1 (TH1)cells (Hsieh C S, et al., 1993, Science. 260: 547-9; Manetti R, et al.,1993, J. Exp. Med. 177: 1199-1204), and forms a link between innateresistance and adaptive immunity. IL-12 has also been shown to inhibitcancer growth via its immuno-modulatory and anti-angiogenesis effects(Brunda M J, et al., 1993, J. Exp. Med. 178: 1223-1230; Noguchi Y, etal., 1996, Proc. Natl. Acad. Sci. U.S.A. 93: 11798-11801; Giordano P N,et al., 2001, J. Exp. Med. 194: 1195-1206; Colombo M P, et al, 2002,Cytokine Growth factor rev. 13: 155-168; Yao L, et al., 2000, Blood 96:1900-1905). IL-12 is produced mainly by dendritic cells (DC) andphagocytes (macrophages and neutrophils) once they are activated byencountering pathogenic bacteria, fingi or intracellular parasites (ReisC, et al., 1997, J. Exp. Med. 186: 1819-1829; Gazzinelli R T, et al.,1994, J. Immunol. 153: 2533-2543; Dalod M, et al., 2002, J. Exp. Med.195: 517-528). The IL-12 receptor (IL-12 R) is expressed mainly byactivated T cells and NK cells (Presky D H, et al., 1996, Proc. Natl.Acad. Sci. U.S.A. 93: 14002-14007; Wu C Y, et al., 1996, Eur J. Immunol.26: 345-50).

Generally the production of IL-12 stimulates the production of INF-γ,which, in turn, enhances the production of IL-12, thus forming apositive feedback loop. In in vitro systems, it has been reported thatIL-12 can synergize with other cytokines (IL-3 and SCF for example) tostimulate the proliferation and differentiation of early hematopoieticprogenitors (Jacobsen S E, et al., 1993, J. Exp Med 2: 413-8; PloemacherR E, et al., 1993, Leukemia 7: 1381-8; Hirao A, et al., 1995, Stem Cells13: 47-53).

However, prior to the present invention, in vivo administration of IL-12was observed to decrease peripheral blood cell counts and bone marrowhematopoiesis (Robertson M J, et al., 1999, Clinical Cancer Research 5:9-16; Lenzi R, et al., 2002, Clinical Cancer Research 8: 3686-95; RyffelB. 1997, Clin Immunol Immunopathol. 83: 18-20; Car B D, et al., 1999,The Toxicol Pathol. 27: 58-63). Using INF-γ receptor knockout mice, Enget al and Car et al demonstrated that high dose IL-12 did not induce thecommonly seen toxicity effect, i.e., there was no inhibition ofhematopoiesis (Eng V M, et al., 1995, J. Exp Med. 181: 1893-8; Car B D,et al., 1995, American Journal of Pathology 147: 1693-707). Thisobservation suggests that the general phenomenon of IL-12 facilitatedenhancement of differentiated hematopoietic cells, as reportedpreviously, may be balanced in vivo by the production of INF-γ, whichacts in a dominant myelo-suppressive fashion.

Without being held to any particular theory, the inventors hypothesizethat, in contrast to previous reports regarding the mechanistic pathwayfor IL-12 mediated proliferation of hematopoietic cells, when thehematopoietic system is compromised, as it is during chemotherapy orradiation therapy, or in the case of certain hematopoietic diseases anddisorders that lead to one or more hematopoietic deficiencies, the 11-12mediated pathway leading to the production of INF-γ may be altered.Thus, besides the low doses used in the examples disclosed herein,another possible mechanism for decreased hematopoietic side effects inembodiments of the invention is that when relatively low dose IL-112 isgiven to a mammal whose hematopoietic system is compromised, theIL-12/INF-γ positive feedback loop may be inhibited. Since INF-γinhibits hematopoiesis and also appears to be the major cytokineresponsible for toxicity, the interruption of INF-γ production may beone of the factors underlying the discovery by the inventors thatadministration of IL-12 provides a hematopoietic protective and recoveryeffect without apparent toxicity.

Therapeutic Methods of the Invention:

The present invention relates to therapeutic methods for treatingdiseases and disorders in which increased amounts of hematopoietic cellsare desirable (e.g., diseases or disorders associated with reducednumbers of one or more hematopoietic cell types or lineages, or diseaseswhere the recommended therapy has associated hematopoietic toxicities,thus leading to reduced numbers of one or more hematopoietic cell typesor lineages, such as cancer) by administration of IL-12, derivatives andanalogs thereof. Thus, embodiments of the invention provide for methodsof alleviating or treating various hematopoietic cell deficiencies,including deficiencies in hematopoietic repopulating cells, progenitorand stem cells, as well as general bone marrow deficiencies, by thedirect administration of IL-12 to a mammal, as disclosed herein.

In the first embodiment of the invention, methods are disclosed fortreating a disease state in a mammal by administering a treatment to themammal that is intended to target the disease state, where the treatmenthas an associated hematopoietic toxicity, in conjunction with theadministration of one or more therapeutically effective dose(s) of IL-12near the time of administration of the treatment. One effect of theadministration of IL-12 to the mammal in this embodiment of theinvention is reduction of the hematopoietic toxicity of the treatment,thus permitting high-dose and dose dense protocols to be utilized indesigned a particular patient's therapeutic regimen.

In a second embodiment of the invention, methods are disclosed foradministration of IL-12 directly to a mammal, preferably a human,suffering from a disease or disorder amenable to treatment by increasingproduction of one or more hematopoietic cell types (e.g., a disease ordisorder associated with a hematopoietic cell deficiency). In a thirdembodiment of the invention, methods of stimulating hematopoiesis in amammal in need comprising administering one or more therapeuticallyeffective dose(s) of IL-12 for a duration to achieve a therapeuticeffect that includes the stimulation of hematopoiesis, wherein thestimulation of hematopoiesis involves the IL-12 facilitatedproliferation of hematopoietic repopulating cells, hematopoieticprogenitor cells or hematopoietic stem cells. In a fourth embodiment ofthe invention, methods are disclosed for bone marrow preservation orrecovery in a mammal by administering one or more therapeuticallyeffective dose(s) of IL-12 to the mammal, without the use of bone marrowcells, hematopoietic progenitor cells or hematopoietic stem cells, for aduration necessary for bone marrow preservation or recovery.

A further aspect of certain embodiments of the invention is that the useof IL-12 as an adjuvant or ancillary therapy to alleviate thehematopoietic toxicities associated with various forms of radiation andchemotherapy, permits high-dose and dose dense treatment protocols to beutilized, and thus, achieve greater rates of remission of the particulardisease state and overall patient survival.

A particular embodiment of the invention provide for methods of treatinga disease state in a mammal. In this embodiment, the disease state canbe any disease state that is treated with either any chemotherapy orradiation therapy, or both. In the invention, the combined use ofchemotherapeutic agents is preferred, as this clinical protocol isgenerally thought to be more therapeutically effective.

These methods generally include administering a treatment to the mammalthat is intended to target the disease state. In this embodiment, thetreatment, which is intended to combat the disease state, also has anassociated hematopoietic toxicity. A second component in this embodimentof the invention includes administering a therapeutically effective doseof IL-12 near the time of administration of the treatment. IL-12 can beadministered at any point in time near the administration of thetreatment that yields the desired therapeutic effect. An overall benefitof practicing this embodiment of the invention is that theadministration of IL-12 to the mammal reduces or decreases thehematopoietic toxicity of the treatment, and as a consequence alters thelimiting toxic dosage of the various treatment modalities.

In these embodiments, the methods generally involve administering aprimary therapeutic to a mammal, along with a secondary therapeutic inthe form of IL-112, where the secondary therapeutic, i.e., IL-12enhances hematopoiesis or improves hematopoietic recovery as comparedwith the administration of the primary therapeutic alone. The preferredmammal in this embodiment of the invention is a human.

The treatment that is intended to target the disease state can be anycurrently practiced therapy, or any therapy to be developed that useseither chemotherapy or radiation therapy, or a combined therapy, toattempt to combat the disease state. In the invention, chemotherapyinvolves the administration of chemical agents, which can be natural orsynthetic agent that are provided in any chemical state, e.g. monomer tohighly polymeric species. In the invention, radiation therapy includesthe administration of relatively high energy wavelengths of light orhigh energy particles to the mammal as the treatment modality. Foreither chemotherapy or radiation therapy, high dose therapeuticapproaches and dose dense protocols as currently practiced or to bedeveloped in the future can be used in the embodiments of the presentinvention.

In general, disorders that can be treated by methods of the inventioninclude, but are not limited to, four broad categories. First arediseases resulting from a failure or dysfunction of normal blood cellproduction and maturation (i.e., aplastic anemia, cytopenias andhypoproliferative stem cell disorders). The second group are neoplastic,malignant diseases in the hematopoietic organs (e.g., leukemia andlymphomas). The third group of disorders comprises those of patientswith a broad spectrum of malignant solid tumors of non-hematopoieticorigin. Induction of hematopoietic cell proliferation in these patientsserves as a bone marrow rescue procedure, without the use of a cellulartransplant, which is provided to a patient as an adjuvant therapy tochemotherapy and/or radiation therapy, including otherwise lethalchemotherapy or radiation therapy and dose dense therapeutic protocols.The fourth group of diseases consists of autoimmune conditions, wherethe enhancement or stimulation of hematopoiesis, leading to increases inhematopoietic cells, can serve as a source of replacement of an abnormalimmune system. Particular diseases and disorders which can be treated byinduction of hematopoietic cell production in vivo are not limited tothose listed in Table 1, and described infra.

TABLE I DISEASES STATES OR DISORDERS WHICH CAN BE TREATED BY INCREASINGHEMATOPOIESIS I. Diseases resulting from a failure or dysfunction ofnormal blood cell production and maturation hypoproliferative stem celldisorders hyperproliefeative stem cell disorders aplastic anemianeutropenia cytopenia anemia pancytopenia agranulocytosisthrombocytopenia red cell aplasia Blackfan-Diamond syndrome due todrugs, radiation, or infection II. Hematopoietic malignancies acutelymphoblastic (lymphocytic) leukemia chronic lymphocytic leukemia acutemyelogenous leukemia chronic myelogenous leukemia acute malignantmyelosclerosis multiple myeloma polycythemia vera agnogenicmyelometaplasia Waldenstrom's macroglobulinemia Hodgkin's lymphomanon-Hodgkin's lymphoma III. Immunosuppression in subjects withmalignant, solid tumors malignant melanoma non-small cell lung cancercarcinoma of the stomach ovarian carcinoma breast carcinoma small celllung carcinoma retinoblastoma testicular carcinoma glioblastomarhabdomyosarcoma neuroblastoma Ewing's sarcoma Lymphoma IV. Autoimmunediseases rheumatoid arthritis diabetes type I chronic hepatitis multiplesclerosis systemic lupus erythematosus V. Genetic (congenital) disordersanemias familial aplastic anemias Fanconi's syndrome Bloom's syndromepure red cell aplasia (PRCA) dyskeratosis congenital Blackfan-Diamondsyndrome congenital dyserythropoietic syndromes I-IV Shwachmann-Diamondsyndrome dihydrofolate reductase deficiencies formamino transferasedeficiency Lesch-Nyhan syndrome congenital spherocytosis congenitalelliptocytosis congenital stomatocytosis congenital Rh null diseaseparoxysmal nocturnal hemoglobinuria G6PD (glucose-6-phosphatedehydrogenase) variants 1, 2, 3 pyruvate kinase deficiency congenitalerythropoietin sensitivity deficiency sickle cell disease and traitthalassemia alpha, beta, gamma met-hemoglobinemia congenital disordersof immunity severe combined immunodeficiency disease (SCID)barelymphocyte syndrome ionophore-responsive combined immunodeficiencycombined immunodeficiency with a capping abnormality nucleosidephosphorylase deficiency granulocyte actin deficiency infantileagranulocytosis Gaucher's disease adenosine deaminase deficiencyKostmann's syndrome reticular dysgenesis congenital leukocytedysfunction syndromes VI. Others osteopetrosis myelosclerosis acquiredhemolytic anemias acquired immunodeficiencies infectious disorderscausing primary or secondary immunodeficiency bacterial infections (e.g.Brucellosis, Listeriosis, tuberculosis, leprosy) parasitic infections(e.g. malaria, Leishmaniasis) fungal infections disorders involvingdisproportions in lymphoid cell sets and impaired immune functions dueto aging phagocyte disorders Kostmann's agranulocytosis chronicgranulomatous disease Chediak-Higachi syndrome Willams-Beuren syndromeneutrophil actin deficiency neutrophil membrane GP-180 deficiencymetabolic storage diseases mucopolysaccharidoses mucolipidosesmiscellaneous disorders involving immune mechanisms Wiskott-AldrichSyndrome alpha 1-antitrypsin deficiency

a) Diseases Resulting from a Failure or Dysfunction of Normal Blood CellProduction and Maturation

In a preferred embodiment of the invention, 1′-12 administration inaccordance with the methods of the invention is used to treat a diseaseresulting from a failure or dysfunction of normal blood cell productionand maturation, such as an aplastic anemia, a cytopenia or ahypoproliferative stem cell disorder. These disorders entail failure ofstem cells in bone marrow to provide normal numbers of functional bloodcells. The aplastic anemias result from the failure of stem cells togive rise to the intermediate and mature forms of red cells, whitecells, and platelets. While red cell production is usually mostseriously affected, a marked decrease in production of other matureblood cell elements is also seen as some anemias specifically affectproduction of white cells and/or platelets. The large majority of theseanemias are acquired during adult life, and do not have any apparentgenetic predisposition. About half of these acquired anemias arise inthe absence of any obvious causative factor such as exposure to poisons,drugs or disease processes that impair stem cell function; these aretermed idiopathic aplastic anemias. The remaining cases are associatedwith exposure to an extremely diverse array of chemicals and drugs andalso occur as the consequence of viral infections and after pregnancy.Other specific types of aplastic anemia are termed agranulocytosis orthrombocytopenia to indicate that the major deficiency lies inparticular white cells or in platelet production, respectively.Additionally, agranulocytosis may be associated with autoimmunesyndromes such as systemic lupus erythematosus (SLE) or with otherinfections, such as neonatal rubella.

In addition, immune deficiencies which are the primary or secondaryresult of infection by pathogenic microorganisms can be treated byadministration of 11-12 according to the methods disclosed in thepresent invention. Microorganisms causing immune deficiencies which maybe treated according to this embodiment of the invention include but arenot limited to gram-negative bacilli such as Brucella or Listeria, themycobacterium which are the etiological agents of tuberculosis or ofHansen's disease (leprosy), parasites such as Plasmodium (theetiological agents of malaria) or Leishmania, and fuigi (such as thosethat cause pneumonia and other lethal infections secondary toimmunodeficiencies) (for a discussion of many of these disorders, seeHarrison's Principles of Internal Medicine, 1970, 6th Edition, Wintrobe,M. M., et al., eds., McGraw-Hill, New York, pp. 798-1044).

b) Treatment of Malignancies

Both chemotherapy and radiation therapy, which are used, either singlyor together, to combat various forms of cancer, and other diseasestates, are toxic to an individual's hematopoietic system. Thus, forindividuals treated with chemotherapy, radiation therapy, or acombination of these two therapeutic modalities, the individual's bloodsupply can be substantially depleted. Moreover, this depletion of theblood supply is generally a limiting factor in the use of chemotherapyand/or radiation therapy to combat various cancers and other diseasestates, and therefore generally precludes the use of high dose or dosedense treatment regimens.

Previously, IL-12 has been reported to play a pivotal role as animmuno-modulator, and has previously been shown to inhibit tumor growthin mice, IL-12 h as been tested in phase I and II human clinical trialsfor its potential to stimulate an immune response in cancer therapy (EngV M et al., Journal of Experimental Medicine 181: 1893-8; Car B D etal., American Journal of Pathology 147: 1693-707). One of the commonside effects of IL-12 therapy, however, is a transient decrease in bloodcell counts: lymphopenia is common (Eng V M et al., Journal ofExperimental Medicine 181: 1893-8; Car B D et al., American Journal ofPathology 147: 1693-707). In animal toxicity studies, lymphopenia isalso a common side effect (Neta R, et al., J. Immunol. 153: 4230-7;Hayes M P et al., Blood 91: 4645-4651).

In contrast to previous studies, however, the inventors have made thediscovery that when IL-12 is administered during certain time “windows”in relation to the time of a primary therapy, such as chemotherapy orradiation therapy, administration of IL-12 increases the nadir of bloodcell counts broadly, i.e., increases blood cell counts, without anyobservable toxic effects of the IL-12 administration.

Thus, the therapeutic methods of the present invention can promotehematopoiesis in general, and in particular, promote hematopoiesis in anindividual who has undergone, is undergoing, or will undergochemotherapy and/or radiation therapy as treatment regimens that targetthe particular malignancies. Thus, in the particular embodiments of theinvention, IL-12 administration is used as an adjuvant or ancillarytherapy to one or more primary therapies implemented near the time ofadministration of the primary therapy. Thus, the present inventionenhances hematopoietic recovery, as well as the general recovery, of asubject undergoing one or more therapies that incidentally decreases theindividual's blood supply. As used herein, the term “undergoing”encompasses the implementation of a primary therapy before, duringand/or after the implementation of the ancillary therapy of the presentinvention.

Among the individual-derived benefits that are a consequence of usingthe methods of the present invention as an adjuvant or ancillary therapyto chemotherapy and/or radiation therapy is a decrease in the toxic sideeffects of these primary therapeutic modalities, as well as enhancedrecovery from these toxic side effects. These toxic side effects includedepletion of one or more blood components of the subject's hematopoieticsystem. A particular individual-derived benefit of administering themethods of the present invention is that more aggressive primarytreatment modalities can be used to combat the targeted disease state.Thus, the use of the methods of the present invention as an adjuvant orancillary therapy allows the primary therapy to be administered in adose dense treatment modality or high dose modality. In turn, thelikelihood of success of the primary therapy is substantially increasedwhen therapeutic compositions including IL-12 are administered as anancillary therapy or combination therapy along with traditionaltherapeutic modalities. Another use of the therapeutic methods of thepresent invention is in the treatment of bone marrow failure resultingfrom certain disease states or is induced by the use of certaintreatment modalities, such as aggressive chemotherapy and/or radiationtherapy.

Hyperproliferative malignant stem cell disorders as well asnon-hematopoietic malignancies can be treated with chemotherapy and/orradiation therapy along with rescue of hematopoietic cells by directadministration IL-12 as disclosed herein. The conditions that can betreated according to the invention include, but are not limited to, theleukemias listed in Table 1 and the solid tumors listed in Table 1.

These malignancies are currently treated by, inter alia, chemotherapyand/or radiation therapy, when feasible, allogeneic bone marrowtransplantation. However, allogeneic HLA identical sibling bone marrowis available only to less than one-third of patients, and this treatmentis associated with transplantation-related complications such asimmunodeficiency and graft versus host disease. Induction ofhematopoietic cell proliferation in vivo via the methods of theinvention permits hematopoietic reconstitution of patients lackingsuitable allogeneic donors, or in the case of an autologous transplant,eliminates the risk of reintroduction of malignant cells. Thus, themethods of the invention can be administered to a patient who hasundergone chemotherapy and/or radiation therapy for treatment of canceror an immunological disorder. Also included in this embodiment of theinvention is the use of administration of IL-12 as an adjuvant therapyto the various therapies used to treat infection, such as HAART therapyand/or radiation therapy, such as total lymphoid irradiation describedbelow.

c) Autoimmune Disorders

Many chronic inflammatory and degenerative diseases are characterized bya continuous immune reaction against the body's own tissues. Suchautoimmune disorders include but are not limited to rheumatoid arthritisand other inflammatory osteopathies, diabetes type I, chronic hepatitis,multiple sclerosis, and systemic lupus erythematosus. Autoimmunedisorders are often treated by lymphoid irradiation. Administration of11-12 as disclosed herein can be valuable to repopulate thehematopoietic system after radiotherapy.

Anti-inflammatory drugs such as steroids retard the inflammatory cellswhich are activated by autoreactive T cells, but do not prevent T cellswhich recognize self-proteins from activating new inflammatory cells. Amore direct approach to treating autoimmune diseases depends oneradication of T cells by irradiation of the lymphoid tissues, andrelying on stem cells from the unirradiated bone marrow to repopulatethe patient's hematopoietic system. The rationale is that the formationof new populations of mature T cells from bone marrow stem cells mayresult in absence of T cells that have reactivity to self-specificantigens. This procedure, called total lymphoid irradiation (TLI), hasbeen used to treat intractable rheumatoid arthritis (Strober, S., etal., 1985, Annals of Internal Medicine 102: 441-449, 450-458). Theseclinical trials showed that in the majority of otherwise intractablecases, joint disease was significantly alleviated for at least 2-3years. However, the major drawback to such treatment is failure of stemcells in the bone marrow of these elderly patients to efficientlyrepopulate the hematopoietic system, resulting in infections andbleeding disorders. Analogous studies have been made of the effects ofTLI as an alternative to cytotoxic drugs for treatment of SLE (Strober,S., et al., 1985, Ann. Internal Med. 102: 450). Studies of the use ofTLI to treat intractable SLE have also shown that this treatmentalleviates disease activity, but is severely limited by failure of bonemarrow stem cells to rapidly and efficiently repopulate thehematopoietic system after irradiation. Thus, the therapeutic methods ofthe invention can be administered to promote proliferation of theremaining hematopoietic cells to increase the success of TLI therapy.

d) Methods of Administration of IL-12

The invention provides methods of treatment by administration to asubject of one or more effective dose(s) of IL-12 for a duration toachieve the desired therapeutic effect. The subject is preferably amammal, including, but not limited to, animals such as cows, pigs,horses, chickens, cats, dogs, etc., and is most preferably human.

Various delivery systems are known and can be used to administer IL-12in accordance with the methods of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing IL-12, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J. Biol. Chem. 262: 4429-4432), construction of nucleic acidcomprising a gene for IL-12 as part of a retroviral or other vector,etc. Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes.

In accordance with the methods of the invention, IL-12 can beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce pharmaceutical compositions comprising IL-12 intothe central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. It may bedesirable to administer the pharmaceutical compositions comprising IL-12locally to the area in need of treatment; this may be achieved, forexample and not by way of limitation, by topical application, byinjection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers.

Other modes of IL-12 administration involve delivery in a vesicle, inparticular a liposome (see Langer, Science 249: 1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

Still other modes of administration of IL-12 involve delivery in acontrolled release system. In certain embodiments, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987);Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med.321: 574 (1989)). Additionally polymeric materials can be used (seeMedical Applications of Controlled Release, Langer and Wise (eds.), CRCPres, Boca Raton, Fla. (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23: 61(1983; see also Levy et al., Science 228: 190 (1985); During et al.,Ann. Neurol. 25: 351 (1989); Howard et al., J. Neurosurg. 71: 105(1989)), or a controlled release system can be placed in proximity ofthe therapeutic target, i.e., the brain, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlledrelease systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

e) Forms and Dosages of IL-12

Suitable dosage forms of IL-12 for use in embodiments of the presentinvention encompass physiologically acceptable carriers that areinherently non-toxic and non-therapeutic. Examples of such carriersinclude ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and PEG. Carriers for topical or gel-based forms of IL-12polypeptides include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, andwood wax alcohols. For all administrations, conventional depot forms aresuitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., supra and Langer, supra, orpoly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate (Sidman et al, supra),non-degradable ethylene-vinyl acetate (Langer et al., supra), degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated IL-12 polypeptidesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

Sustained-release IL-12 containing compositions also include liposomallyentrapped polypeptides. Liposomes containing a IL-12 polypeptide areprepared by methods known in the art, such as described in Eppstein etal., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045and 4,544,545. Ordinarily, the liposomes are the small (about 200-800Angstroms) unilamelar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal Wnt polypeptide therapy. Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556.

For the treatment of disease, the appropriate dosage of a IL-12polypeptide will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, previous therapy, thepatient's clinical history and response to the IL-12 therapeutic methodsdisclosed herein, and the discretion of the attending physician. Inaccordance with the invention, IL-12 is suitably administered to thepatient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 10 ng/kg to2000 ng/kg of IL-12 is an initial candidate dosage for administration tothe patient, whether, for example, by one or more separateadministrations, or by continuous infusion. Humans can safely tolerate arepeated dosages of about 500 ng/kg, but single dosages of up to about200 ng/kg should not produce toxic side effects. For example, the dosemay be the same as that for other cytokines such as G-CSF, GM-CSF andEPO. For repeated administrations over several days or longer, dependingon the condition, the treatment is sustained until a desired suppressionof disease symptoms occurs. However, other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques and assays.

IL-12 may be administered along with other cytokines, either by directco-administration or sequential administration. When one or morecytokines are co-administered with IL-12, lesser doses of IL-12 may beemployed. Suitable doses of other cytokines, i.e. other than IL-12, arefrom about 1 ug/kg to about 15 mg/kg of cytokine. For example, the dosemay be the same as that for other cytokines such as G-CSF, GM-CSF andEPO. The other cytokine(s) may be administered prior to, simultaneouslywith, or following administration of IL-12. The cytokine(s) and IL-12may be combined to form a pharmaceutically composition for simultaneousadministration to the mammal. In certain embodiments, the amounts ofIL-12 and cytokine are such that a synergistic repopulation of bloodcells (or synergistic increase in proliferation and/or differentiationof hematopoietic cells) occurs in the mammal upon administration ofIL-12 and other cytokine thereto. In other words, the coordinated actionof the two or more agents (i.e. the 11-12 and one or more cytokine(s))with respect to repopulation of blood cells (orproliferation/differentiation of hematopoietic cells) is greater thanthe sum of the individual effects of these molecules.

Therapeutic formulations of IL-12 are prepared for storage by mixingIL-12 having the desired degree of purity with optional physiologicallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, 16th edition, Osol, A., Ed., (1980)), in theform of lyophilized cake or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counter-ions such as sodium; and/or non-ionic surfactantssuch as Tween®, Pluronics™ or polyethylene glycol (PEG).

IL-12 also may be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles, and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

IL-12 to be used for in vivo administration must be sterile. This isreadily accomplished by filtration through sterile filtration membranes,prior to or following lyophilization and reconstitution. IL-12ordinarily will be stored in lyophilized form or in solution.Therapeutic IL-12 compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

When applied topically, 11-12 is suitably combined with otheringredients, such as carriers and/or adjuvants. There are no limitationson the nature of such other ingredients, except that they must bephysiologically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. Examples of suitable vehicles includeointments, creams, gels, or suspensions, with or without purifiedcollagen. The compositions also may be impregnated into transdermalpatches, plasters, and bandages, preferably in liquid or semi-liquidform.

For obtaining a gel formulation, IL-12 formulated in a liquidcomposition may be mixed with an effective amount of a water-solublepolysaccharide or synthetic polymer such as PEG to form a gel of theproper viscosity to be applied topically. The polysaccharide that may beused includes, for example, cellulose derivatives such as etherifiedcellulose derivatives, including alkyl celluloses, hydroxyalkylcelluloses, and alkylhydroxyalkyl celluloses, for example,methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxypropyl methylcellulose, and hydroxypropyl cellulose; starch andfractionated starch; agar; alginic acid and alginates; gum arabic;pullullan; agarose; carrageenan; dextrans; dextrins; fructans; inulin;mannans; xylans; arabinans; chitosans; glycogens; glucans; and syntheticbiopolymers; as well as gums such as xanthan gum; guar gum; locust beangum; gum arabic; tragacanth gum; and karaya gum; and derivatives andmixtures thereof. The preferred gelling agent herein is one that isinert to biological systems, nontoxic, simple to prepare, and not toorunny or viscous, and will not destabilize the IL-12 molecule heldwithin it.

Preferably the polysaccharide is an etherified cellulose derivative,more preferably one that is well defined, purified, and listed in USP,e.g., methylcellulose and the hydroxyalkyl cellulose derivatives, suchas hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethylcellulose. Most preferred herein is methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight PEGs to obtain the proper viscosity. Forexample, a mixture of a PEG of molecular weight 400-600 with one ofmolecular weight 1500 would be effective for this purpose when mixed inthe proper ratio to obtain a paste.

The term “water soluble” as applied to the polysaccharides and PEGs ismeant to include colloidal solutions and dispersions. In general, thesolubility of the cellulose derivatives is determined by the degree ofsubstitution of ether groups, and the stabilizing derivatives usefulherein should have a sufficient quantity of such ether groups peranhydroglucose unit in the cellulose chain to render the derivativeswater soluble. A degree of ether substitution of at least 0.35 ethergroups per anhydroglucose unit is generally sufficient. Additionally,the cellulose derivatives may be in the form of alkali metal salts, forexample, the Li, Na, K, or Cs salts.

If methylcellulose is employed in the gel, preferably it comprises about2-5%, more preferably about 3%, of the gel and IL-12 is present in anamount of about 300-1000 mg per ml of gel.

An effective amount of IL-12 to be employed therapeutically will depend,for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer IL-12 until a dosage is reachedthat achieves the desired effect. A typical daily dosage for systemictreatment might range from about 10 ng/kg to up to 2000 ng/kg or more,depending on the factors mentioned above. As an alternative generalproposition, the IL-12 receptor is formulated and delivered to thetarget site or tissue at a dosage capable of establishing in the tissuean IL-12 level greater than about 0.1 ng/cc up to a maximum dose that isefficacious but not unduly toxic. This intra-tissue concentration shouldbe maintained if possible by the administration regime, including bycontinuous infusion, sustained release, topical application, orinjection at empirically determined frequencies. The progress of thistherapy is easily monitored by conventional assays.

EXAMPLES Example 1

A study was conducted to assess the ability of IL-112 administration toprotect, or rescue, a mammal from lethal ionizing radiation and theresults are presented in FIG. 1. Mice received recombinant murine IL-12(100 ng/mouse (which is about 5 ug/kg on average), intravenousinjection) before (24 hours) or after (1 hour) lethal dose radiation(1000 rad). Mice in control group received equal volume of PBS buffer.Survival rate were analyzed by Kaplan-Meier method and p value wascalculated using Log Rank Test. The survival difference between IL-12treatment (▴, n=38 or Δ, n=35) and control (□, n=62) is statisticallysignificant (p<0.001). The survival difference between mice who receivedIL-12 before (▴) radiation and the control (□, n=62), and the survivaldifference between mice who received IL-12 after (Δ) radiation and thecontrol (□, n=62) are also both statistically significant (p<0.05).

Example 2

A study was conducted to assess a low dose IL-12 can effectively protecta mammal from lethal dose ionizing radiation. To determine the minimumeffective radioprotective dose of IL-12, different dose of IL-112 wereadministered to lethally irradiated animals before (24 hours before) orpost (within one hour) lethal dose ionizing radiation. The best survivalrate was obtained at dose of 5 ug/kg, in both before and post radiationadministration. Higher dose (up to 50 ug/kg) did not yield greatersurvival. The dose of 5 ug/Kg can be converted to approximately 400ng/kg as the appropriate human dose, using the general rule where thehuman dose is 1/12 of the murine dose. As shown in FIG. 1, whenadministrated at 100 ng/mouse (5 ug/Kg), 95% (administrated 24 hoursbefore radiation) and 75% (administrated post radiation) animals showedlong-term survival (more than one year) respectively, while all thecontrol animals died within 24 days after receiving lethal doseradiation. IL-12 treated animals showed significant better survival(p<0.001) as compared with animals in control group. Interestingly, thedifference before the animals received IL-12 before radiation andanimals received IL-12 after radiation as compared with the control isalso significant (p<0.05).

Example 3

IL-12 was administrated to mice at different doses 24 hours beforeradiation to determine if there is a dose dependent relationship betweenthe concentration of IL-12 administration and survival from lethalirradiation. As shown in the Table 2, the optimal radioprotective dosein mice is about 100 ng/mouse or 5 ug/kg. This dose can be converted toabout 400 ng/kg in humans (as the human dose is generally 1/12 of themurine dose).

TABLE 2 Radioprotection of IL-12 is dose dependent IL-12 dose SurvivesSurvival rate (ng/mouse) Total mice (30 days) (%) 0 5 0 0 5 5 0 0 10 5 00 25 5 2 40 50 5 3 60 100 5 4 80 200 5 3 60IL-12 was administrated at different dose 24 hours before radiation. Asshown in the table, the optimal radioprotective dose is 100 ng/mouse.

Example 4

To further explore the relationship of IL-12 hematopoietic rescue effectas it may relate to the time of administration, IL-12 was administratedto lethally irradiated animals at different time before and after theradiation. The following time points were tested: −48, −36, −24, and −12hours before radiation, or +1, +12, +24, and +36 hours post radiation.As shown in Table 3, when IL-12 administrated before radiation, the bestresults come from at −24 hours with all the animals survived (10 out of10); while given post radiation, +1 hour gives the best rescue (4 out of5 mice survived). Other injection time showed no (0% survival rate at−48, −36, +12 and +36 hours injection time for tested 5 animals in eachgroup) or little (20% survival rate at −12 and +24 hours injection timefor tested 5 animals).

TABLE 3 Radioprotective effects of IL-12 is injection-time dependentInjection time Injection time before IR (hrs) after IR (hrs) control 4836 24 12 1 12 24 36 Total 10 10 10 10 10 5 5 10 5 mice Survives 0 0 0 102 4 0 2 0 Survival 0 0 0 100 20 80 0 20 0 rate (%)IL-12 was administrated at different time before or post lethal doseradiation and the survival of the animals were observed daily. As shownin table 2, the best administration time for radioprotection is 24 hoursbefore or 1 hour post radiation. IR: lethal irradiation

Example 5

A study was conducted to determine if the radioprotective effect ofIL-12 administration is specific and INF-γ independent. To elucidate ifthis radioprotective effect is a common property of IL-12-relatedcytokines or if the radioprotective effect is IL-12 specific, thefollowing cytokines were tested in the radioprotection assay: 1) IL-23,a heterodimer (p19+p40), which shares the same subunit p40 with IL-12(p35+p40); IL-23 also shares the same receptor subunit, namely IL-12Rb,with IL-12; moreover, similar IL-12, the binding of IL-23 with itsreceptor result INF-γ production; 2) IL-18, like IL-12, can induce therelease of interferon γ; 3) IL-2, which can synergistically functionwith IL-12 in immune response and increases the expression of IL-12receptor in T cells; 4) GM-CSF, a cytokine to stimulate G and M cellproduction. As shown in Table 4, IL-12 is the only cytokine tested thatcan protect mice from lethal dose radiation (4 out of 5 mice survivedwhen IL-12 is administrated post radiation). Also, an assessment wasmade to determine if there was a synergistic effect between GM-CSF andIL-12. No synergistic effect was found. Thus, the radioprotectivefunction of IL-12 is specific and is interferon γ independent.

TABLE 4 Radioprotective effects of different cytokines Survival Totalmice Survives rate (%) Control 5 0 0 IL-2 5 0 0 IL-18 4 0 0 IL-23 5 0 0GM-CSF 4 0 0 IL-12 5 4 80 IL-12 + GM-CSF 5 4 80Different cytokines were administrated to mice 1 hour post lethal doseradiation. The survival rates were observed by checking the animalsurvival daily. Although both IL-18 and IL-23 can induce INF-γproduction, there was no radioprotective effect from these cytokines.This results suggested the radioprotective effect was IL-12 specific andINF-γ independent. Since GM-CSF only did not show any radioprotectiveeffect and nor additive effect when used with IL-12 together, IL-12 onlyis sufficient to protect the animals from lethal dose radiation. *: IL:interleukin; GM-CSF: granulocyte-macrophage colony-stimulating factor.

Example 6

A study was conducted to determine the effects of IL-12 administrationand radiation on bone marrow and the intestinal tract. The majorquestion to be answered in this study is whether IL-12 can protect bonemarrow from the harmful effects of radiation without sensitizing theintestinal tract. The conclusion of the study is that IL-12 protectsbone marrow from ionizing radiation, but does not sensitize theintestinal tract to the ionizing radiation.

To determine if IL-112 sensitizes intestinal tract to ionizingradiation, two different doses of IL-12 and radiation were used inradioprotection assay, namely 5 ug/kg and 50 ug/kg of IL-12 and twodifferent doses of radiation were also used. Subsequent to theadministration of IL-12 and radiation, both bone marrow and the smallintestine were removed from mice who received either 5 ug/kg or 50 ug/kgof IL-12 and a lethal dose radiation for histology study. The lethaldoses were either 1000 rad or 1600 rad. With 1000 rad, or 1600 rad), anda dose of 5 ug/kg IL-12, there is no significant difference for smallintestine in control mice and IL-12 treated mice: the small intestinewas intact in terms of shape and number of villa and crypt in the micereceived 1000 rad radiation (FIG. 3), while there is lethal GI tractdamage in the mice received 1600 rad radiation (mice both in controlgroup and IL-12 treated group). However, bone marrow presented differentprofiles as shown in FIG. 2: Il-12 treated animals (5 ug/kg) showedprotected marrow with significant higher cellularity at day 1 (controlvs. IL-12 administration). There is no significant difference of bonemarrow histological structure between mice in control and in IL-12treated group at day 7 and day 10 post radiation (data not shown),though the bone marrow cellularity of IL-12 treated mice is better thancontrol mice (data not shown). However, colonies were observed in IL-12treated mice bone marrow at day 12 post radiation. By day 14, there isfull recovery of bone marrow cellularity in IL-12 treated mice.Different from IL-12 treated group, the mice in control group did notshow signs of bone marrow recovery all the time points studied.

Example 7

A study was conducted to show whether IL-12 administration promotesmulti-lineage blood cell recovery from the effects of ionizingradiation. The peripheral blood cell counts drop is an index for damageof ionizing radiation on hematopoietic system. Since IL-12 demonstratedradioprotective effect from ionizing radiation, peripheral blood cellrecovery was monitored in both lethal and sublethal dose radiation.

After lethal dose radiation (10 Gy), the full blood cell count droppedto lowest point for both IL-12 treated and non-treated mice. However,11-12 treated mice start to recover at day 14 post radiation while thecontrol animals died out. The IL-12 treated animals reach to normalblood cell count at about 30 days post radiation, as shown in FIG. 6. Itis noteworthy that the recovery is multi-lineage, including white bloodcell, red blood cell and platelets. This result is expected to havesignificant clinical value as disclosed above.

As shown in FIG. 4, sublethal radiation (500 rad), IL-12 accelerates theblood cell count recovery in treated mice as compared with controlanimals. Here too, the recovery is broad range and encompasses all bloodlineages and blood cell types. Especially at day 5 and 8 post radiation,the red blood cell, platelet, white blood cell and monocyte from IL-12treated mice are statistically higher as compared with control mice(p<0.05). This effect is somewhat better when IL-12 was administrated 24hours before radiation than IL-12 administrated after radiation.

Example 8

A study was conducted to assess whether IL-12 can protect progenitorcells in bone marrow from a lethal dose radiation

To determine the bone marrow cell subsets that are protected by IL-12administration for the rescue phenomena, CFC and CFU-S₁₂ assay wereperformed at different time post lethal dose radiation. Bone marrowcells were recovered from IL-12 treated or control animals immediately(day 0), or at day 7, 9, 10, 12, 14 and 21 post radiation for CFC andCFU-S₁₂ assay. If progenitor cells are protected from IL-12 treatment(IL-12 was administered in this study at 24 hours before radiation), CFCand CFU-S₁₂ colonies should be detected when cells were removed at day0. However, bone marrow cells isolated at day 0, 7 post radiation giverise no CFC nor CFU-S₁₂ colonies in both IL-12 treated and controlanimals. At day 10 post radiation, the bone marrow cells isolated fromIL-12 treated mice start to give rise of CFU-S₁₂ but no detectable CFC,which start to appear from IL-112 treated bone marrow isolated 12 dayspost radiation and fully recovered from bone marrow cells isolated 14days post radiation, as shown in FIGS. 5A and 5B. These results suggestthat IL-12 does not directly protect bone marrow progenitor andshort-term HSCs from lethal dose radiation. One explanation for thisresult is that there may be no CFC and CFU-S₁₂ colony forming cell leftfrom lethal dose radiation in IL-12 treated animal bone marrow. TheCFU-S₁₂ colony forming cells and CFC cells at day 10 and day 14 postradiation may be derived from long-term repopulating stem cells rescuedby IL-12 treatment.

The experimental details are as follows. At different time post lethaldose radiation, bone marrow cells were isolated from mice who receivedIL-12 or PBS buffer for colony forming cell assay (CFC assay) (5A),colony forming units spleen day 12 assay (CFU-S₁₂) (5B-5C), and bonemarrow transplantation (BMT, 5D-F). Legend: D0: immediately afterirradiation; IR, irradiation. BM, bone marrow. CFU-S₁₂, colony formingunits—spleen12.

FIG. 5A: Isolated bone marrow cells were plated in methycellulose platesfor 12 days to detect colony formation. Bone marrow cells from normalmice were used as control for the assay system. There is no detectableCFC activity the first 9 days in both control and IL-12 treated mice. Byday 12, there start to show low level CFC cell activity in IL-12 treatedmice. The full recovery of CFC activity was observed at about 14 dayspost radiation in IL-12 treated mice. In contract, there is no recoveryof CFC cell activity from control mice bone marrow cells.

FIG. 5B-C: Isolated bone marrow cells from IL-12 treated or control micewere transplanted to second lethally irradiated mice. 12 days posttransplantation, the recipient mice were sacrificed and spleens wereremoved, fixed to count for colony formation. Cells isolated the first 7days of both IL-12 treated and control mice did not give rise ofCFU-S₁₂. However, a full recovered CFU-S₁₂ activity was observed fromcells isolated 10 days post radiation in IL-12 treated mice. FIG. 5Bshows the summary of 2 experiments with total of 6 mice; FIG. 5C showsthe spleens isolated from mice received IL-12 or PBS buffer (control)bone marrow cells.

FIG. 5D-F: Secondary bone marrow transplantation was employed to detectif IL-12 can protect long-term repopulating HSC. Six month after lethaldose radiation and IL-12 rescue, bone marrow cells from these mice (Ly5.2) were isolated and transplanted to secondary lethally irradiatedmice (Ly 5.1). Four months after transplantation, donor cells (Ly5.2)were examined in recipient's peripheral blood. As shown in FIG. 5F,about 30% repopulated blood cells are of donor origin and donor cellderived T/B cell (FIG. 5E) and M/G (FIG. 5D) cell can be detectedindicating that IL-12 protects long-term repopulating cells.

The following gives some further experimental details. Six months afterthe rescue from lethal dose radiation (IL-12 treated mice, C57BL/Ly5.2),whole bone marrow cells were isolated and 1×10⁶ cells were transplantedto secondary lethally irradiated mice (C57BL/Ly5.1). 4 months aftertransplantation, blood cells were collected and stained with anti-Ly5.2and anti-Mac-1/anti-Gr-1 antibodies (5D), and anti-Ly5.2 andanti-CD3/anti-B220 antibodies (5E) and anti-Ly5.2 antibody (5F). IL-12rescued bone marrow cells gave rise to both myeloid lineage cells (5D,cells showed double positive of Ly5.2 and Mac-1/Gr-1 antigens); andlymphoid lineage cells (5E, cells showed double positive of Ly5.2 andCD3/B220 antigens). There were about 28±1% blood cells derived fromIL-12 rescued bone marrow donor cells (5F). D: days after irradiation.

Example 9

A study was conducted to determine whether IL-12 administrationstimulates bone marrow cell proliferation. The results of the studyreveal that IL-12 promotes bone marrow cells into cycling andproliferation as compared with controls.

It is believed that cells at S phase are more resistant to radiation.Both Brd-U incooperation and cell cycling analysis were performed todetermine if IL-12 can stimulate bone marrow cell proliferation. InIL-12 treated mice, there are more bone marrow cells in S phase comparedwith control animals (12% vs 10%, p<0.05). Furthermore, there are moreBrdU positive cells in IL-12 treated bone marrow compared with controlanimals (17% vs 8%, p<0.05).

These results are shown in FIG. 7. 24 hours post IL-12 administration(10 ng/mouse), bone marrow cells were isolated for BrdU incooperationassay (FIG. 7A). Statistical analysis showed IL-12 treated bone marrowcontains higher levels of BrdU positive cells compared with controlmouse (FIG. 7B, p<0.01, n=6). Cell cycle analysis were performed withPropidium Iodide staining method which showed significant increased cellnumber at S phase (C, p<0.05, n=6).

Example 10

A study was conducted to assess whether IL-12 administration protectsSca-1+ cells from radiation induced apoptosis. The Sca-1+ is a markerfor hematopoietic repopulating cell or stem/progenitor cells. 24 hourspost IL-12 administration, mice received lethal dose radiation (10 Gy).7 hours post radiation, mice were sacrificed and bone marrow cells wereisolated to detect Annexin V (an indication of cell apoptosis) and Sca-1antigen (stem cell marker). As shown in FIG. 8, compared with PBStreated control mice, the AnnexinV⁻ (negative for Annexin V)/Sca-1⁺cells in IL-12 treated mice are significantly increased (19.2±3.7% vs9.7±1.5%, p<0.01). The proportion of AnnexinV^(−(negative))/Sca-1⁺ cellsin total Sca-1⁺ cells is significantly higher for IL-12 treated animalsas compared with control mice (54±5% vs 44±5%, p<0.05).

Example 11

A study was conducted to determine whether IL-12 administration promotesmulti-lineage blood cell recovery when used in conjunction with achemotherapeutic drug. In this example, use of IL-12 is as an adjuvantor ancillary therapy to the primary therapy of chemotherapy. IL-12 wasadministrated at different time (36 hrs before or 12 hrs afterchemotherapy). Mice in this study received a relatively high dose ofchemotherapeutic drug Cytoxan (e.g., 300 mg/kg). At different timepost-cytoxan treatment, peripheral blood was collected via tail vein forblood cell count study. (Mascot). These data are shown in FIG. 9. FIG.9A white blood cell count; B: red blood cell count; C: platelet count.With limited mouse number (n=5), IL-12 treated mice showed better bloodcell recovery.

Materials and Methods Mice and Cytokines

All mice used in the above disclosed experiments were purchased fromJackson Labs (Bar Harbor, Me.). The specific mice used were C57BL/6Jstrain female mice were, generally 6 to 8 weeks old. Mice were handledand managed according to the protocol approved by the University ofSouthern California Animal Care and Use Committee. Recombinant murineinterleukin 12 (IL-12) was purchased either from R&D Systems, Inc(Minneapolis, Minn.) or PeproTech Inc (Rocky Hill, N.J.) and wasdissolved in phosphate buffered saline (PBS), pH 7.4 at 100 ng/ul stockconcentration according to the manufacturer's recommendation, and storedat −70° C. Other cytokines or chemokines were bought from R&D Systems,Inc.

Radioprotection Assay

Mice were provided with acid water for one week before theradioprotection assay. Cytokines, diluted in phosphate buffered saline(PBS), pH 7.4, were intravenously injected into mice before or aftertotal body lethal irradiation (TBI) at the specified times. Controlgroup mice were injected with PBS buffer. For TBI, mice were eitherexposed twice to ionizing irradiation (γ-ray from Cesium 137) at 500 raddose with a 3 hour interval, or one time at a dose of 1000 rad in aGammacell 40 machine from Atomic Energy of Canada LTD (Kanafa, Ontario,Canada). The γ-ray exposure rate is 1 Gy/min. This radiation dose willcause all control group mice to die within 30 days after exposure. Afterirradiation, mice were given water containing antibiotics. For thesurvival curve, the Kaplan-Meier method was used. Each experiment wasrepeated three or more times.

For peripheral blood cell counts, 6-8 week old female C57BL/6J micereceived an i.v. injection of IL-12 before or after lethal (1000 rad) orsub-lethal (500 rad) irradiation. 10 ul blood was taken from the tail todo blood cell count analyses in a MASCOT Multispecies Hematology Systems(CDC Technologies, Oxford, Conn.) at different days after irradiation.Each group had at least 3 mice.

Cell Cycling and Proliferation Assay

For the cell cycling assay, IL-12 (100 ng/mouse) or PBS wasintravenously injected into mice. 24 hours after injection, whole bonemarrow cells were flushed from femurs and treated with lysis buffer todestroy red blood cells. After washing in PBS and fixing in −20° C. pureethanol for at least 1 hour, about 5×10⁵ bone marrow cells were treatedwith RNase A (20 ng/ul final concentration) at 37° C. for 30 minutes,then stained with propidium iodide (100 ng/ul final concentration) fromSigma (St Louis, Mo.) at room temperature for 15 to 30 minutes and thenprocessed directly for cell cycling analysis using fluorescenceactivated cell sorting (FACS).

For the BrdU assay, IL-12 (10 ng/mouse) or PBS was intravenouslyinjected into mice. 21 hours after injection, mice were i.p. injectedwith 5-Bromo-2′-deoxyuridine (BrdU from Sigma) at 50 mg/kg dosage. 3hours after BrdU injection, mice were sacrificed and whole bone marrowcells were isolated from femurs and treated with lysis buffer to destroyred blood cells. Bone marrow cells were dispersed on a slide by cytospinfor the proliferation assay using BrdU immunohistochemistry system kitpurchased from Oncogene Research Products (Boston, Mass.)

Pathology of Bone Marrow, Spleen and Small Intestine

Mice were treated with IL-12 (100 ng/mouse) or PBS 24 hours beforereceiving lethal irradiation. At different days after lethalirradiation, femurs, spleens and small intestines were removed and fixedin 10% formalin buffer for 24 hours. Subsequently, bone marrow wasdecalcified in Rapid Decalcifier for about 30 minutes. Samples andtissues then were embedded in TissuePrep 2 paraffin wax formicro-section at 5 μm for routine Hematoxylin and Eosin staining. Somesmall intestines were treated with PAS (Periodic Acid Schiff) staining.

IL-12 Radioprotective Effects on Long-Term Hematopoietic Stem Cells

Ly5.2 strain mice rescued by IL-112 treatment before or after lethalirradiation were sacrificed after 6 months. Whole bone marrow cells fromthese mice as donor cells (1×10⁶ Ly5.2 cells per recipient mouse or5×10⁵ Ly5.2 cells with 5×10⁵ Ly5.1 competitor cells per recipient) weretransplanted into Ly5.1 strain mice which received total body lethalirradiation. 4 months after the transplantation, peripheral blood cellswere analyzed for Ly5.2 cells (anti-Ly5.2 antibody conjugated with FITC)with T cell and B cell markers (anti-CD3 and anti-B220 antibodyconjugated with PE) and with macrophage and granulocyte cell markers(anti-CD11b and anti-Gr1 antibody conjugated with PE) using FACS. Allantibodies were purchased from BD Pharmingen (San Diego, Calif.)

Colony-Forming Units (CFU) Assay and CFU-Spleen₁₂ Assay

24 hours before lethal irradiation, mice were i.v. treated with IL-12(100 ng/mouse) and bone marrow cells were isolated from these mice atdifferent days after lethal irradiation. For CFU assay, 2×10⁵ bonemarrow cells were thoroughly mixed into 1 ml methylcellulose mediumMethCult GF M3434 from StemCell Technologies Inc. (Vancouver, BC) andcultured into 35 mm dishes. Each sample was duplicated. After 12 daysculture, colonies (>50 cells) were counted under microscope.

For the CFU-spleen1₂ assay, 2×10⁵ bone marrow cells as donors weretransplanted into recipient mice which received lethal irradiation.Cells from one donor mice were transplanted into three recipient mice.12 days after transplantation, recipient mice were sacrificed andspleens were fixed in Tellyesniczky's solution (90 ml of 70% ethanol, 5ml of glacial acetic acid, and 5 ml of 100% formalin). Spleen colonieswere counted.

Although the present invention has been discussed in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references and patents cited herein are incorporated byreference in their entirety.

1-118. (canceled)
 119. A method for protecting or restoringhematopoiesis or enhancing peripheral blood cell counts in a mammalhaving at least one solid tumor, wherein the method comprises: (a)administering a treatment to the mammal, wherein the treatment targetsthe solid tumor and has an associated hematopoietic toxicity, andwherein the treatment is selected from the group consisting of radiationtherapy or a combination of chemotherapy and radiation therapy; and (b)administering one or more therapeutically effective dose(s) ofinterleukin-12 (IL-12) near the time of administration of the treatment,wherein the administration of IL-12 to the mammal reduces thehematopoietic toxicity of the treatment.
 120. The method of claim 119,wherein the method results in an increased remission of at least onesolid tumor, as compared to the treatment intended to target the diseasestate alone.
 121. The method of claim 119, wherein the mammal is ahuman.
 122. The method of claim 119, wherein the dose of IL-12 isselected from the group consisting of 2000 ng/kg, less than about 2000ng/kg, about 500 ng/kg, less than about 500 ng/kg, about 400 ng/kg,about 200 ng/kg, less than about 100 ng/kg, about 10 ng/kg, and lessthan about 10 ng/kg.
 123. The method of claim 119, wherein: (a) thetreatment comprises one or more high dose treatment modalities; (b) thetreatment is administered in a dose dense treatment regimen; or (c) acombination thereof.
 124. The method of claim 119, wherein the radiationtherapy comprises a near-lethal dose of radiation.
 125. The method ofclaim 119, wherein the radiation therapy comprises a sub-lethal dose ofradiation.
 126. The method of claim 119, wherein; (a) the administrationof IL-12 results in protection of bone marrow cells from the associatedhematopoietic toxicity of the treatment; (b) the IL-12 administrationresults in radioprotection of bone marrow cells; (c) the treatmentcomprises chemotherapy and administration of IL-12 results inchemoprotection of bone marrow cells; or (d) any combination thereof.127. The method of claim 126, wherein the bone marrow cells comprisehematopoietic repopulating cells, hematopoietic stem cells,hematopoeitic progenitor cells, or any combination thereof.
 128. Themethod of claim 119, wherein the treatment leads to a deficiency in oneor more hematopoietic cell types or lineages and the administration ofIL-12 ameloriates the deficiency.
 129. The method of claim 119, whereinthe deficiency is selected from the group consisting of: (a) a generaldeficiency in hematopoiesis in the mammal; (b) a deficiency in one ormore specific hematopoietic cell lineages; or (c) a combination thereof.130. The method of claim 129, wherein the deficiency in one or morespecific hematopoietic cell lineages is selected from the groupconsisting of lymphopenia, myelopenia, leukopenia, neutropenia,erythropenia, megakaryopenia, and any combination thereof.
 131. Themethod of claim 119, wherein the treatment comprises chemotherapy andthe chemotherapy treatment comprises administration of more than onechemotherapy.
 132. The method of claim 119, wherein one or moretherapeutically effective dose(s) of IL-12 is administered at varioustime intervals before, before and after, or after the administration ofthe treatment.
 133. The method of claim 119, wherein: (a) IL-12 isadministered before the chemotherapy; (b) IL-12 is administered afterthe chemotherapy; (c) IL-12 is administered before and after thechemotherapy; (d) IL-12 is administered before the radiation therapy;(e) IL-12 is administered after the radiation therapy; (f) IL-12 isadministered before and after the radiation therapy; or (g) anycombination thereof.
 134. The method of claim 119, wherein the IL-12 isadministered at about 24 hours before radiation.
 135. The method ofclaim 119, wherein the IL-12 is administered at about 1 hour or morefollowing radiation.
 136. The method of claim 119, wherein the solidtumor is associated with a disease state selected from the groupconsisting of cancer, breast cancer, lung cancer, prostate cancer,ovarian cancer, hematopoietic cell cancers, leukemias, lymphomas,hyperproliferative malignant stem cell disorders, non-hematopoieticmalignancies, malignant melanoma, non-small cell lung cancer, carcinomaof the stomach, ovarian carcinoma, breast carcinoma, small cell lungcarcinoma, retinoblastoma, testicular carcinoma, glioblastoma,rhabdomyosarcoma, neuroblastoma, and Ewing's sarcoma.
 137. The method ofclaim 119, wherein a cytokine is administered with the IL-12, eithersequentially or by direct co-administration.
 138. The method of claim137, wherein the cytokine is selected from the group consisting ofG-CSF, GM-CSF, EPO, IL-23, IL-18, and IL-2.